Bottom Line:
We also described the production of highly angiogenic EPC-comparable cells from pluripotent embryonic stem cells (ESCs) by direct differentiation using EC-conditioned medium (ECCM).ECCM-treated ESC-derived progenitor cells exhibited angiogenic potential, demonstrated by in vitro tubule formation, and endothelial-specific gene expression equivalent to natural EPCs.We concluded the effect of EPCs is cumulative and beneficial, relying on upregulation of the angiogenic activity of transplanted cells combined with an increase in proliferative cell number to produce significant effects upon transplantation.

Background: Endothelial progenitor cells (EPCs) are implicated in a range of pathological conditions, suggesting a natural therapeutic role for EPCs in angiogenesis. However, current angiogenic therapies involving EPC transplantation are inefficient due to rejection of donor EPCs. One solution is to derive an expanded population of EPCs from stem cells in vitro, to be re-introduced as a therapeutic transplant. To demonstrate the therapeutic potential of EPCs we performed in vitro transplantation of EPCs into endothelial cell (EC) tubules using a gel-based tubule formation assay. We also described the production of highly angiogenic EPC-comparable cells from pluripotent embryonic stem cells (ESCs) by direct differentiation using EC-conditioned medium (ECCM).

Results: The effect on tubule complexity and longevity varied with transplantation quantity: significant effects were observed when tubules were transplanted with a quantity of EPCs equivalent to 50% of the number of ECs originally seeded on to the assay gel but not with 10% EPC transplantation. Gene expression of the endothelial markers VEGFR2, VE-cadherin and CD31, determined by qPCR, also changed dynamically during transplantation. ECCM-treated ESC-derived progenitor cells exhibited angiogenic potential, demonstrated by in vitro tubule formation, and endothelial-specific gene expression equivalent to natural EPCs.

Conclusions: We concluded the effect of EPCs is cumulative and beneficial, relying on upregulation of the angiogenic activity of transplanted cells combined with an increase in proliferative cell number to produce significant effects upon transplantation. Furthermore, EPCs derived from ESCs may be developed for use as a rapidly-expandable alternative for angiogenic transplantation therapy.

Mentions:
To determine whether EPC transplantation could enhance the longevity of existing EC tubules, we added EPCs to ECMatrix gel containing branching ECs that had been in culture for 5 h. Transplantation was performed with EPCs equal to 50% or 10% of the original number of ECs. Following transplantation with 10% equivalent EPCs, the greatest number of N1 nodes occurred at 2 h, decreasing by 6 h (Figure 6a). However, there was no significant decrease in N1 nodes between 6 h and 8 h. A progressive decrease in N1 nodes was then seen between 10 h and 12 h, and by 14 h no N1 nodes were present. The maximum number of N2 and N3 nodes occurred at 4 h (1 h prior to transplantation), whilst N4 nodes were maximal at 6 h (1 h after transplantation) and N5+ nodes were only evident at 6 h. Nevertheless, in the 50% EPC-equivalent transplantation assay, it is evident that the number of N1 nodes decreased progressively between 2 h and 8 h (Figure 6b). However, an increase in N1 nodes was seen by 10 h (P < 0.01), and between 10 h and 14 h the mean number of N1 nodes did not change significantly. The greatest number of N2 nodes was recorded at 4 h (1 h prior to transplantation) although N3 nodes were maximal at 8 h (3 h after transplantation). However, the mean number of N4 nodes, first evident at 4 h, increased significantly from 4 h to 6 h (P < 0.05), then decreased progressively between 8 h and 12 h. N5+ nodes were first identified at 6 h, with no significant difference between 6 h and 10 h with a slight reduction between 10 h and 14 h, whilst a significant decrease was observed at 14 h (P < 0.05).

Mentions:
To determine whether EPC transplantation could enhance the longevity of existing EC tubules, we added EPCs to ECMatrix gel containing branching ECs that had been in culture for 5 h. Transplantation was performed with EPCs equal to 50% or 10% of the original number of ECs. Following transplantation with 10% equivalent EPCs, the greatest number of N1 nodes occurred at 2 h, decreasing by 6 h (Figure 6a). However, there was no significant decrease in N1 nodes between 6 h and 8 h. A progressive decrease in N1 nodes was then seen between 10 h and 12 h, and by 14 h no N1 nodes were present. The maximum number of N2 and N3 nodes occurred at 4 h (1 h prior to transplantation), whilst N4 nodes were maximal at 6 h (1 h after transplantation) and N5+ nodes were only evident at 6 h. Nevertheless, in the 50% EPC-equivalent transplantation assay, it is evident that the number of N1 nodes decreased progressively between 2 h and 8 h (Figure 6b). However, an increase in N1 nodes was seen by 10 h (P < 0.01), and between 10 h and 14 h the mean number of N1 nodes did not change significantly. The greatest number of N2 nodes was recorded at 4 h (1 h prior to transplantation) although N3 nodes were maximal at 8 h (3 h after transplantation). However, the mean number of N4 nodes, first evident at 4 h, increased significantly from 4 h to 6 h (P < 0.05), then decreased progressively between 8 h and 12 h. N5+ nodes were first identified at 6 h, with no significant difference between 6 h and 10 h with a slight reduction between 10 h and 14 h, whilst a significant decrease was observed at 14 h (P < 0.05).

Bottom Line:
We also described the production of highly angiogenic EPC-comparable cells from pluripotent embryonic stem cells (ESCs) by direct differentiation using EC-conditioned medium (ECCM).ECCM-treated ESC-derived progenitor cells exhibited angiogenic potential, demonstrated by in vitro tubule formation, and endothelial-specific gene expression equivalent to natural EPCs.We concluded the effect of EPCs is cumulative and beneficial, relying on upregulation of the angiogenic activity of transplanted cells combined with an increase in proliferative cell number to produce significant effects upon transplantation.

Background: Endothelial progenitor cells (EPCs) are implicated in a range of pathological conditions, suggesting a natural therapeutic role for EPCs in angiogenesis. However, current angiogenic therapies involving EPC transplantation are inefficient due to rejection of donor EPCs. One solution is to derive an expanded population of EPCs from stem cells in vitro, to be re-introduced as a therapeutic transplant. To demonstrate the therapeutic potential of EPCs we performed in vitro transplantation of EPCs into endothelial cell (EC) tubules using a gel-based tubule formation assay. We also described the production of highly angiogenic EPC-comparable cells from pluripotent embryonic stem cells (ESCs) by direct differentiation using EC-conditioned medium (ECCM).

Results: The effect on tubule complexity and longevity varied with transplantation quantity: significant effects were observed when tubules were transplanted with a quantity of EPCs equivalent to 50% of the number of ECs originally seeded on to the assay gel but not with 10% EPC transplantation. Gene expression of the endothelial markers VEGFR2, VE-cadherin and CD31, determined by qPCR, also changed dynamically during transplantation. ECCM-treated ESC-derived progenitor cells exhibited angiogenic potential, demonstrated by in vitro tubule formation, and endothelial-specific gene expression equivalent to natural EPCs.

Conclusions: We concluded the effect of EPCs is cumulative and beneficial, relying on upregulation of the angiogenic activity of transplanted cells combined with an increase in proliferative cell number to produce significant effects upon transplantation. Furthermore, EPCs derived from ESCs may be developed for use as a rapidly-expandable alternative for angiogenic transplantation therapy.